Aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing.
Aptamers, like peptides generated by phage display or monoclonal antibodies (“mAbs”), are capable of specifically binding to selected targets and modulating the target's activity or binding interactions, e.g., through binding aptamers may block their target's ability to function. Discovered by an in vitro selection process from pools of random sequence oligonucleotides, aptamers have been generated for over 130 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size (20-45 nucleotides), binds its target with nanomolar to sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteins from the same gene family). A series of structural studies have shown that aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarities, hydrophobic contacts, steric exclusion) that drive affinity and specificity in antibody-antigen complexes.
Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties, in addition, they offer specific competitive advantages over antibodies and other protein biology, for example:
1) Speed and control. Aptamers are produced by an entirely in vitro process, allowing for the rapid generation of initial leads, including therapeutic leads. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled and allows the generation of leads, including leads against both toxic and non-immunogenic targets.
2) Toxicity and Immunogenicity. Aptamers as a class have demonstrated therapeutically acceptable toxicity and lack of immunogenicity. Whereas the efficacy of many monoclonal antibodies can be severely limited by immune response to antibodies themselves, it is extremely difficult to elicit antibodies to aptamers most likely because aptamers cannot be presented by T-cells via the MHC and the immune response is generally trained not to recognize nucleic acid fragments.
3) Administration. Whereas most currently approved antibody therapeutics are administered by intravenous infusion (typically over 2-4 hours), aptamers can be administered by subcutaneous injection (aptamer bioavailability via subcutaneous administration is >80% in monkey studies (Tucker et ah, J. Chromatography B. 732: 203-212, 1999)). With good solubility (>150 mg/mL) and comparatively low molecular weight (aptamer: 10-50 kDa; antibody: 150 kDa), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 mL. In addition, the small size of aptamers allows them to penetrate into areas of conformational constrictions that do not allow for antibodies or antibody fragments to penetrate, presenting yet another advantage of aptamer-based therapeutics or prophylaxis.
4) Scalability and cost. Therapeutic aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Whereas difficulties in scaling production are currently limiting the availability of some biologies and the capital cost of a large-scale protein production plant is enormous, a single large-scale oligonucleotide synthesizer can produce upwards of 100 kg/year and requires a relatively modest initial investment. The current cost of goods for aptamer synthesis at the kilogram scale is estimated at $500/g, comparable to that for highly optimized antibodies. Continuing improvements in process development are expected to lower the cost of goods to <$100/g in five years.
5) Stability. Therapeutic aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to factors such as heat and denaturants and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders. [0010] Furthermore, the aptamer discovery process readily permits lead modification, such as aptamer sequence optimization and the minimization of aptamer length [Conrad et al. 1996, Eaton et al. 1997]. Additionally, 2′ modifications such as 2′-fluoro and 2′-O-Me may be utilized for stabilization against nucleases without compromising the aptamer binding interaction with the target. See e.g. Lin et al. Nucleic Acids Res. 22, 5229-5234 (1994); Jellinek et [alpha]l, Biochemistry 1995, 34, 11363-1137; Lin et [alpha]l, Nucleic Acids Res., 1994, 22, 5229-5234; Kubik et [alpha]l., J. Immunol., 1997, 159(1), 259-267; and Pagratis et [alpha]l., Nat. Biotechnol., 1997, 1, 68-73.
Severe malaria is almost exclusively caused by P. falciparum infection and usually arises 6-14 days after infection. Consequences of severe malaria include coma and death if untreated—young children and pregnant women are especially vulnerable. Splenomegaly (enlarged spleen), severe headache, cerebral ischemia, hepatomegaly (enlarged liver), hypoglycemia, and hemoglobinuria with renal failure may occur. Renal failure may cause blackwater fever, where hemoglobin from lysed red blood cells leaks into the urine. Severe malaria can progress extremely rapidly and cause death within hours or days. In the most severe cases of the disease fatality rates can exceed 20%, even with intensive care and treatment. In endemic areas, treatment is often less satisfactory and the overall fatality rate for all cases of malaria can be as high as one in ten. Over the longer term, developmental impairments have been documented in children who have suffered episodes of severe malaria.
Plasmodium falciparum is the causative agent of severe malaria in humans. Millions of people worldwide are infected every year by P. falciparum and more than one million die, most of them small children in sub-Saharan Africa. The choice of drugs for malaria treatment has in the past primarily been quinine and chloroquine and since the 1960s sulfadoxine/pyrimethamine (SP). Unfortunately parasite resistance towards all these drugs has been documented in endemic regions. The most efficient drug used today as a first-line treatment is artemisinin and its derivates. Although no clinical resistance towards artemisinin has been demonstrated, there are indications of a developed in vitro resistance towards the drug in P. falciparum. Non-sterile immunity against severe malaria in residents in endemic regions has been described. This indicates the existence of antigenic homogeneity in the parasites causing severe malaria.
The protein known to be responsible for severe cerebral malaria by the process of rosetting and endothelial adherence is the erythrocyte membrane protein 1 (PfEMP1) expressed on the surface of the infected erythrocyte. Exposed on the infected erythrocyte it binds to a number of human cell surface receptors such as heparin sulphate, ICAM-1, CD36, CSA, enabling the parasite to adhere to the endothelial linings of small blood vessels (cytoadherence) as well as to non-infected erythrocytes (rosetting), thus preventing spleenic clearance from the bloodstream. Such sequestered parasites cause considerable obstruction to tissue perfusion.
PfEMP1 consist mainly of duffy binding ligand domains (DBL's) and cysteine rich inter domain regions (CIDR's), and the number of domains and size of the protein varies depending on which of the 60 var-genes is expressed. The fact that the parasite regularly changes the expressed var-gene and thus generating antigenic variation of the infected erythrocyte surface facilitates parasite avoidance of host immune system. Certain sequence conservation is though expected due to the adhesive function of this protein. The virulence-associated phenotype, rosetting, is mediated by the N-terminal Duffy-binding-like domain (DBL1alfa) which has a high degree of sequence conservation among the PfEMP1 domains. The definitive structure of DBL1α is not known though extensive modelling on domains with similar structure has been performed. This makes DBL1 an attractive candidate for the development of novel drugs against severe malaria. Attempts of antibody recognition of the structural conserved epitopes in DBL1α have been made without greater success due to the fact that the conserved regions are somewhat masked by the variable ones making them inaccessible to the comparatively large antibody.
Chen, Q. et al, Vaccine 22, (2004) p. 2701-2712 discloses that Immunization with PfEMP1-DBL1α generates antibodies that disrupt rosettes and protect against sequestration of Plasmodium falciparum-infected erythrocytes. The use of the PfEMP1-DBL1α protein to produce antibodies is a secondary therapeutic treatment, while the market asks for a direct therapeutic agent and method.
Moll, K. et al, Inf. Imm. vol. 75(1), (January 2007), p. 211-219 discloses generation of cross-protective antibodies against Plasmodium falciparum sequestration by immunization with an erythrocyte membrane protein 1-duffy binding like 1α domain. Also this work deals with the use of the PfEMP1-DBL1α protein to produce antibodies is a secondary therapeutic treatment, while the market asks for a direct therapeutic agent and method.
Based on these characteristics the present inventors designed a Systematic Evolution of Ligand by Exponential Enrichment (SELEX) protocol, which could be designed to allow the selection of RNA aptamers to bind with high affinity and specificity to the structurally conserved parts of DBL1α.
Ulrich, H. et al, J. Biol. Chem., vol. 277 (2002), p. 20756-20762 describes the SELEX method for in vitro selection of RNA aptamers that bind to cell adhesion receptors Trypanosoma cruzi and inhibit cell invasion.
A similar strategy has been reported for other pathogenic parasites where aptamers have successfully been selected against virulent surface proteins. In one reported case high affinity RNA aptamers were selected against the variable surface glycoprotein (VSG) of Trypanosoma brucei that proved to be capable of directing antibodies to the surface of live trypanosomes. The other reported study used a different approach. The selection was not performed using expressed surface proteins from Trypanosoma cruzi but using a displacement technique with 4 different human surface receptors (Ulrich, H. et al, supra).
Trypanosoma cruzi causes heart problems in the chronic stage, whereby treatment involves managing the clinical manifestations of the disease. For example, pacemakers and medications for irregular heartbeats may be life saving for some patients with chronic cardiac disease, while surgery may be required for megaintestine. The disease cannot be cured in this phase, however. Chronic heart disease caused by Chagas disease is now a common reason for heart transplantation surgery. Until recently, however, Chagas disease was considered a contraindication for the procedure, since the heart damage could recur as the parasite was expected to seize the opportunity provided by the immunosuppression that follows surgery.
Aptamers have been shown to present the same high specificity and affinity for their targets as antibodies. In addition to efficient binding, aptamers also display an inhibitory activity of their targets. The SELEX method is based on an iterative process of repeating steps of in vitro selection cycles where the initial DNA/RNA library of 1014-1015 different molecules is reduced to a smaller pool of approximately 100 different molecules that have high affinity towards the target in question.
Lee, J. F., et al, Curr Opi. Chem. Biol. (2006), 10:282-289 is a review over aptamer therapeutics advance. Thereby anti-HIV-1, anti-VEGF, anti-RET, anti-theophylline, and anti-tenascin-C aptamers are discussed.
Hjalmarsson, K. et al, FOI-R-1216-SE (ISSN 1650-1942) discusses Aptamers—Future tools for diagnostics and therapy, and is in particular related to the SELEX-methodoly. An overview of different aptamers and their optional use is given.
Göringer, H. U., et al, Int. J. Parasit. 33 (2003), 1309-1317 discloses in vitro selection of high-affinity nucleic acid ligands to parasite target molecules, and discusses malaria, whereby the authors concluded primarily that the process of development has been slow because of the random screening methods of components have not been successful at identifying anti-parasitic compounds. The authors concentrate of discussing the SELEX protocol. Although malaria is a first parasite to be mentioned there is no hint that it adverse effects can be treated using an aptamer, but the authors discuss Trypanosoma bruzei causing sleeping sickness.
Normark, J. et al, PNAS, (2007) vol. 104, p. 15835-15840 discloses that PfEMP1-DBL1α amino acid motifs are preent in severe disease states of Plasmodium falciparium malaria. The paper does not discuss aptamers as potential agents in the treatment of severe malaria caused by Plasmodium falciparium but discusses raising of antibodies as a possible regime.
Krause, D. R. et al, Inf. Imm. vol 75, (2007) p. 5967-5973 discloses antibody response against Plasmodium falciparum and discusses that serum from a volunteer inhibits resetting. The authors state that “the diversity of this protein (PfEMP1) within and between parasites combined with antigenic switching between variants makes studying the development of PfEMP1-specific immunity difficult.
WO 2004/080420 to Mota et al discloses in general terms a methods for preventing or inhibiting the activity of malaria in vivo by administering an antimalarial agent to a mammal in need thereof, and indicates a number of different pathways to become interfered. In particular the disclosure discusses MET inhibition, MET being the receptor for Hepatocyte Growth Factor. The disclosure even mentions aptamers without any specification. In general the disclosure is very conceptual without delivering any specific solutions, but only thoughts about possible routes for treating malaria.
Jayasena, S. D. Clin. Chem. 45:9 (1999) p. 1628-1650 discloses Aptamers as an emerging class of molecules that rival antibodies in diagnostics. However, the disclosure gives no indication of therapeutic aptamers for treating malaria.
The present inventors further show that these specific high affinity binding aptamers are able to bind to PfEMP1 on the surface of live parasite and, lastly, that they have the capacity to disrupt rosettes. Herein it will be demonstrated specific binding of a set of aptamers that have an effect in vitro in formation of rosettes. It is further proposed to use these aptamers to diagnose the presence or not of severe malaria, i.e., to discriminate between light (or rather less severe forms) and severe forms of malaria.